SUMMARY Brush border microvilli play essential roles in the processing and uptake of nutrients, and defense against pathogenic microbes and toxic compounds that accumulate in the gut lumen. The destruction or malformation of microvilli contributes to numerous intestinal pathologies, including illness caused by attaching and effacing (A/E) pathogens such as Enterohemorrhagic E. coli O157:H7 (EHEC) and microvillus inclusion disease. Despite functioning at a critical physiological interface in the gut, we know little about how microvilli are assembled during the maturation of enterocytes that occurs as these cells exit the crypt. In this proposal, we will begin to elucidate the molecular machinery that drives the growth of microvilli during enterocyte differentiation. We recently discovered the Inverse Bin-Amphiphysin-Rvs (I-BAR) domain containing protein Insulin Receptor Tyrosine Kinase Substrate (IRTKS) as a novel regulator of microvillar growth. BAR family proteins are known drive and/or stabilize membrane curvature; I-BAR domains specifically interact with membranes exhibiting outward curvature, like that generated during microvillar protrusion. Using super- resolution microscopy, we found that IRTKS exhibits striking localization to the distal tips of microvilli, where the growing ends of actin filaments are found. Based on this and other preliminary findings, we propose that during enterocyte differentiation, IRTKS is recruited to regions of active brush border assembly to drive the elongation of nascent microvilli. To test this hypothesis we propose to: (1) elucidate the function of IRTKS during enterocyte differentiation in vivo, (2) define the mechanism underpinning IRTKS-induced microvillar growth, and (3) identify upstream factors that regulate IRTKS localization and activity. These studies will employ a combination of live imaging, state-of-the-art super-resolution microscopy, and new forms of electron microscopy (EM) to test our central hypothesis in cell culture, enteroid, and mouse models. Successful completion of these studies will lead to new paradigms for understanding intestinal epithelial morphogenesis and disease.